Optimized Expression and Purification of Outer Membrane Protein 25 (Omp25) of Brucella melitensis

Our previous immunoproteomic studies on patients with acute brucellosis identified outer membrane protein 25 (Omp25) of Brucella melitensis as one of the immunogenic antigens, indicating its potential to be used as a diagnostic marker and a tool to differentiate acute and chronic human brucellosis. Expression and purification of Omp25 are technically challenging using conventional approaches owing to its membrane association. Existing expression and extraction protocols frequently lead to host cell toxicity, low protein yields, and poor reproducibility. To address these limitations, we developed a modified approach for the expression and purification of recombinant Omp25 protein that is standardized and reproducible and yields protein efficiently. The truncated omp25 gene is cloned and expressed in Escherichia coli BL21 (DE3) cells, and induction is carried out at 28°C for 6 hr. The expressed recombinant Omp25 protein is predominantly found in the insoluble fraction, so it is extracted under denaturing conditions, purified by Ni-NTA affinity chromatography, refolded by stepwise dialysis, and desalted prior to characterization. In our hands, this set of protocols yielded 47.75 mg purified, high-quality recombinant Omp25 protein per liter of bacterial culture. SDS-PAGE analysis demonstrated a single predominant protein band at ∼24.3 kDa with no detectable contaminating protein bands, indicating successful purification. recombinant Omp25-based indirect ELISA confirmed that the refolded protein retained its antigenic properties, exhibiting significantly higher reactivity with sera from culture-confirmed brucellosis patients than with healthy control sera (p < 0.001). This optimized set of protocols provides a reproducible workflow for the production of highly purified recombinant Omp25 protein and offers quantitative performance metrics that facilitate reproducibility and support this protein's application in immunological and diagnostic studies. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and extraction of Brucella melitensis Omp25 protein from Escherichia coli BL21 (DE3) cells Basic Protocol 2: Purification of recombinant Omp25 protein Basic Protocol 3: Refolding and desalting of recombinant Omp25 protein.